Despite many therapeutic advances, early detection of malignancy has great potential as a means of affecting outcome and survival of cancer patients. All too often the disease is too far advanced and therapeutic options are limited. Earlier diagnosis and treatment can affect outcome as evidenced by screening Papanicolou smears for squamous cell carcinoma of the cervix [Lunt (1984) Obstet. Gynecol. 63:708-713].
The development of an assay with similar potential for early detection of other malignancies would be of considerable aid to clinical oncologists. Recently, investigators have identified several substances that are produced by tumor cells for use as diagnostic markers of cancer. These include the germ cell markers human choriogonadotropin and .alpha.-fetoprotein, which are very specific markers, and less sensitive and specific markers such as carcinoembryonic antigen (CEA), CA-12.5, CA-19.9, CA-15.3, prostate specific antigen (PSA), and others. Recent reviews describe the use of these and other tumor markers in diagnostic and therapeutic applications [Pohl et al. (1983) Cancer Detect. Prevent. 6:7-20; Kluvins et al. (1983) Ann. Clin. Lab. Sci. 13:275-280; Sultzeanu et al. (1985) Adv. Cancer Res. 44:1-42; Virji et al. (1988) Cancer 38:105-126; and Sikorska et al. (1988) Cancer Detect. Prevent. 12:321-355].
The cysteine protease, cancer procoagulant (CP), E.C. 3.4.22.26, has been isolated and purified [Gordon et al. (1975) Thrombos. Res. 6:127-138; Gordon et al. (1981) J. Clin. Invest. 67:1665-1671; and Falanga et al. (1985) Biochemistry 24:5558-5567] from neoplastic tissue as well as amnion-chorion tissue. It was found that CP activity existed in the extracts of malignant cells, but not in extracts from normal tissue or serum-free media from normal cells in tissue culture [Gordon et al. (1978) Cancer Res. 38:2467-2472; Gordon et al. (1979) J. Natl. Cancer Inst. 62:773-776; Gordon et al. (1982) Thrombos. Res. 26:379-387; and Gordon et al. (1989) Thrombos. Res. 56:431-440]. Enzymatic and immunological characterization of extracts of malignant and benign melanocytic tissue revealed CP in the melanoma extracts and not in the benign nevi extracts [Donati et al. (1986) Cancer Res. 46:6471-6474]. In acute nonlymphocytic leukemia, CP was identified in most of the cytological subtypes but not in control mononuclear cells from either peripheral blood or bone marrow aspirates [Falanga et al. (1988) Blood 71:870-875]. In addition, a virtually identical procoagulant protein has been purified from human amnion-chorion tissue (fetal origin), implying that CP may be an oncofetal protein [Gordon et al. (1985) Blood 66:1261-1265; and Falanga et al. (1985) Biochem. Biophys. Acta 831:161-165].
Initially, CP was assayed with a one-stage and, later, with a two-stage clotting assays [Gordon et al. (1981) J. Clin. Invest. 67:1665-1671; Falanga et al. (1985) Biochemistry 24:5558-5567; and Gordon et al. (1985) Blood 66:1261-1265]. A direct chromogenic assay for CP was developed by Moore [(1992) Biochem. Biophys. Res. Com. 184:819-824]. This assay used the chromogenic substrate, Chromozym TH, which is very specific for thrombin and, therefore, the assay had low specificity and sensitivity. Also, this assay necessitated the use of very pure CP, without traces of coagulation factors or other proteinases.
"Sandwich" enzyme-linked immunosorbent assays (ELISA) are very sensitive and specific because of the high degree of specificity of antibodies for their antigens. An ELISA assay was developed and used to assay CP [Gordon U.S. Pat. No. 4,943,524, issued Jul. 24, 1990, and Gordon et al. (1990a) Cancer Res. 50:6229-6234]. CP measurements with ELISA assays in the serum of cancer patients and of noncancerous control individuals led to the characterization of CP as a tumor marker.
In studies described by Gordon et al. (1990b) Cancer Res. 50:6229-6234, there was a substantial percentage of false-negative cancer patient sera in both the gastrointestinal and lung cancer groups of patients with late stage cancer. It was noted that several of these "false negative" patients were terminal at the time the blood samples were obtained and that they died from their malignancy shortly thereafter. Blood samples of these cancer patients suggested the presence of endogenous anti-CP antibodies. Evidence was also obtained [Meilicki et al. (1990) Int. J. Cancer 45:125-126] for a decreased production of CP activity in the serum of rats with late stage epithelioma, suggesting that CP production in late stage cancers may decrease. Further, it was suggested that in the false negative samples CP may be associated with a serum protein or other substance produced by the tumor that binds CP, blocks the epitope and inhibits the immunoreactivity of CP in the assay.